Laser scanning confocal microscopy (LSCM) has been widely employed in our laboratory for structural and functional analysis of clinical specimens and cultured cell lines. This powerful technology has several advantages over conventional epi-fluorescence microscopy, including analysis of fluorescent labeled thick specimens without physical sectioning, free out-of-focus blur for thin or thick specimens, and capacity for complex three-dimensional (XYZ) architectural data by integrating serial optical sections taken at different focal planes which create an animated sequence to reveal latent features of specimens. We use the laser scanning system Fluoview 1000 (Olympus) to study the localization and co-localization of tight junction components in periodontal disease lesions. Live cell imaging is used to measure the diffusion patterns of fluorescent molecules in response to a bacterial virulence factor, the gingipain haemagglutinin domain, that attacks epithelial integrity. 3-D reconstruction is used to observe tooth structure and pulp tissues from carious teeth. This technology is also used to assess penetration of novel antimicrobial drugs designed to kill intracellular pathogens. This indicates that confocal microscopy is powerful for gaining advanced knowledge of structural and functional cellular properties in dental research. Future development in confocal microscopy applications will be of great interest and benefit to biomedical research.